Are cell redox or lactate dehydrogenase kinetics responsible for the absence of gluconeogenesis from lactate in sea raven,hepatocytes?

Previous studies have reported very low rates of gluconeogenesis from lactate in sea raven (Hemitripterus americanus) hepatocytes compared to other teleosts studied. This study examines whether hepatic cell redox or lactate dehydrogenase (LDH) characteristics may explain this observation. Sea raven hepatic optimal LDH activities (pyruvate reductase direction) were more than 40 times less compared with rainbow trout liver values (40 vs 1914 μmol·min⁻¹·g⁻¹ protein). The Km(lactate) was 9.24 and 0.86 mM for sea raven and trout hepatic LDH, but the Km(pyruvate) was similar between the two species (0.11 and 0.21 mM, respectively). These results suggested that sea raven liver LDH did not favour lactate use and was more indicative of the mammalian M-isozyme. Gel electrophoresis showed a predominant intermediate isozyme, with a small amount of the M-type LDH. Phosphoenolpyruvate carboxykinase (PEPCK) was localized to the mitochondrial compartment, while there was no apparent mitochondrial glutamate-oxaloacetate transaminase (GOT) activity. No in vitro lactate flux to glucose was found in untreated, 10 mM ethanol-treated, or 3 mM NH₄Cl-treated sea raven hepatocytes, although CO₂ production from lactate was decreased by ethanol and increased by NH₄Cl. These results provide evidence that cell redox does not limit gluconeogenesis from lactate, while low activities and the kinetic characteristics of LDH may partially explain the low lactate gluconeogenesis reported in sea raven hepatocytes. To whom correspondence should be addressed at University of Ottawa.     

Effects of dietary ascorbic acid/iron ratio on some production traits, lipid peroxide state and amount/activity of the glutathione redox system in African catfish Clarias gariepinus (Burchell) fingerlings

African catfish Clarias gariepinus (Burchell) fingerlings (3.16–3.92 g initial body weight) were investigated for 30 days in four different groups using different amounts of l ‐ascorbic acid (AA) and iron (supplied as FeC₆H₅O₇) in their feedings. Diet 1 (control): no addition of AA or iron; diet 2 (H‐AA/FE): high (600 mg kg^?1) AA and low (218 mg kg^?1) iron; diet 3 (H‐HE/AA): high (364 mg kg^?1) iron and low (200 mg kg^?1) AA; and an unfed group, which was investigated only for 15 days due to high mortality. The live weight gain, feed intake, specific growth rate (SGR; % body weight day^?1) and feed conversion rate (FCR) were measured or calculated. At the end of the experimental period, the whole body content of AA, iron, reduced glutathione (GSH), glutathione disulphide (GSSG) and malondialdehyde (MDA), as well as the glutathione peroxidase (GSHPx) activity, were measured. The production traits did not differ significantly as a result of the different AA and iron contents of the feed. AA content increased significantly in all the groups as compared with the initial value, except in the unfed group. The difference between the treated groups as compared with the control, with regard to the two AA/iron treated groups, was also significant. The iron content in the fish body increased significantly compared with the initial value, except in the unfed group. The difference compared with the control was significant only in the H‐FE/AA group. The difference between the groups that consumed low and high iron content diets was also significant. The GSH and GSSG content, as well as the GSH/GSSG ratio and GSHPx activity of the fish body, did not differ significantly as compared with the initial value or with the control. The lipid peroxide status, as measured by the MDA content, did not differ significantly either as an effect of the AA and iron supplementation, but decreased as an effect of ageing and starvation. It may be concluded that, under the present experimental conditions, the C. gariepinus fingerling tissue stores of AA and/or iron increased as a result of feed supplementation, but without altering the actual lipid peroxide status and the amount/activity of the glutathione redox system.     

Volume regulation in glutathione-treated brook trout (Salvelinus fontinalis) erythrocytes

Brook trout erythrocytes that were washed with and suspended in Ringer's solution with reduced glutathione (1.0 mM) maintained steady state cell volume for up to 24h, while those without the thiol-protective agent steadily shrank. Changes in cell volume (measured as packed cell volume, PCV) were evoked by acidic media (Ringer's at pH 6.8), hypoosmotic solutions (or both) and intracellular K⁺ and Cl⁻ concentrations were monitored over 4h. Acid-swollen cells failed to volume regulate or release K⁺ but had significantly elevated intracellular Cl⁻ Osmotically-swollen cells at pH 7.8 but not at pH 6.8 underwent regulatory volume decrease (RVD) and returned to initial levels in 2h, accompanied by release of K⁺ and Cl⁻ In contrast, osmotically-shrunken cells did not show regulatory volume increase. The regulatory volume decrease and concomitant K⁺ release were dependent on Cl⁻ implying a direct or indirect coupling of K⁺ to Cl⁻ transport in volume regulation. RVD was partially blocked by 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS, 0.1 mM), an anion exchange blocker, but was unaffected by amiloride (1.0 mM) which blocks Na⁺/H⁺ exchange. Amiloride and DIDS prevented the swelling response to low pH but had no effect on control cells, suggesting involvement of Na⁺/H⁺ and Cl⁻/HCO₃ ⁻ exchanges in acid-induced cell swelling. Quinine (1.0 mM) a known blocker of K⁺ channels, exacerbated the osmotically-induced swelling but had little effect on the subsequent RVD and release of KCl. The results suggest that low extracellular pH inhibits neutral C⁻-dependent K⁺ release and the resultant regulatory volume decrease in osmotically-swollen cells.     

Effects of in vitro exposure to ozone and/or hyperoxia on superoxide dismutase, catalase, glutathione and lipid peroxidation in red blood cells and plasma of rainbow trout, Oncorhynchus mykiss (Walbaum)

In aquaculture, ozone is used as a disinfectant. In its production, extensive amounts of oxygen are formed resulting in hyperoxic conditions in culture units. Both ozone and hyperoxia have the potential to be toxic via pro‐oxidant mechanisms and to activate antioxidant defence systems in cultured species. To eliminate systemic effects, blood of rainbow trout, Oncorhynchus mykiss (Walbaum), was exposed in vitro for 5 min to ozone/hyperoxia or hyperoxia, and changes in antioxidant defences and lipid peroxidation were measured after exposure. Ozone exposure caused severe damage in red blood cells (rbc) detected as increased lipid peroxidation and oxidized glutathione (GSSG) levels in both plasma and rbc. Oxygen exposure alone increased intracellular lipid peroxidation and GSSG levels 10 min after exposure and was not evident in the plasma at any time. Ozone, but not oxygen exposure, decreased reduced glutathione (GSH) levels in plasma, and the changes were negatively correlated with increased lipid peroxidation in rbc, indicating that extracellular GSH has a dynamic role in the protection of rbc from direct oxidation by ozone. Both ozone and hyperoxic conditions increased superoxide dismutase (SOD) activity in rbc 3 and 6 h after exposure. In contrast, catalase activity was only increased 10 min after oxygen exposure, suggesting other catalase activation mechanisms rather than enzyme induction. The recovery of lipid peroxidation and GSSG levels in rbc after hyperoxia, but not ozone exposure, indicated a capacity to defend against hyperoxia‐produced oxidative damage, but an overwhelming of antioxidant defences by ozone in rainbow trout rbc in vitro.     

Influence of dietary n-3 fatty acids on the desaturation and elongation of [1-14C] 18:2 n-6 and [1-14C] 18:3 n-3 in Atlantic salmon hepatocytes

Atlantic salmon (Salmo salar) were fed diets containing fish oil supplemented with 22:6n-3 (FO diet) or linseed oil supplemented with 20:5n-3 (LO diet) for 6 months. The effects of these diets, both containing about 36% n-3 fatty acids, on the esterification, desaturation and elongation of [1-¹⁴C] 18:2n-6 and [1-¹⁴C] 18:3n-3 were investigated in isolated hepatocytes. The percentages of radioactivity which was esterified from [1-¹⁴C] 18:2n-6 or [1-¹⁴C]18:3n-3 into total lipids, were approximately 20% lower in hepatocytes from fish fed the FO diet than in hepatocytes from fish fed the LO diet. The percentages of radioactivity esterified in both groups were further reduced when 0.1 mM unlabelled 22:6n-3 was added to the incubation. The percentage of desaturation and elongation products formed from [1-¹⁴C] 18:2n-6 was twice as high in hepatocytes from salmon fed the FO diet as it was in hepatocytes from fish fed the LO diet. The ratio of 18:2n-6 to 18:3n-3 was five times higher in the FO diet, and this probably promoted the conversion of 18:2n-6 to longer chain n-6 fatty acids. When 0.1mM unlabelled 22:6n-3 was added to the incubation medium, the percentages of desaturation and elongation products formed were unchanged. Thus, a high level of 22:6n-3 in the diet is apparently not inhibiting the conversion of 18:2n-6 to 20:4n-6, as long as the amount of 18:2n-6 present is substantially higher than that of 18:3n-3. No desaturation and elongation products were recovered from the phospholipids of hepatocytes incubated with [1-¹⁴C] 18:3n-3 in any of the groups. However, the `dead end' elongation product 20:3n-3 was found in the triacylglycerol fraction, and the percentage of this fatty acid increased when 22:6n-3 was added to the incubation medium.     

L‐carnitine exerts a cytoprotective effect against H2O2‐induced oxidative stress in the fathead minnow muscle cell line

This study was conducted to investigate the protective effect of L‐carnitine (LC) against H₂O₂‐induced oxidative stress in the fathead minnow muscle cell line (FHM). The FHM cells were stimulated with 1 mM H₂O₂ for 1 h after LC pre‐treatment, and the cell viability and the activity and mRNA relative expression of antioxidant enzyme were measured to assess the antioxidant properties of LC. The results showed that the toxic effect of H₂O₂ on the viability of FHM cells was both dose‐ and time‐dependent. Furthermore, the viability of the 0.01–1 LC mM groups was significantly higher than those of the 1 mM H₂O₂ group. L‐carnitine protected the cells from H₂O₂‐induced oxidative damage, which was demonstrated by a significant reduction in the malondialdehyde and reactive oxygen species levels and increases in the intracellular total glutathione levels and the activities of total superoxide dismutase, catalase, glutathione peroxidase (GPx) and gamma‐glutamyl‐cysteine synthetase (γ‐GCS) in FHM cells pre‐treated with LC for 6 h compared with the 1 mM H₂O₂ group. In addition, the mRNA relative expression levels of the γ‐GCS catalytic subunit and nuclear factor nuclear factor erythroid 2‐related factor 2 were significantly higher than those of the 1 mM H₂O₂ group. It could be concluded that LC exerts a beneficial antioxidant effect against oxidative stress induced by H₂O₂ in FHM cells and that the appropriate treatment is 0.1–1 mM for 6 h in this study.     

Hepatoprotective and antioxidant effects of Glycyrrhiza glabra extract against carbon tetrachloride (CCl4)-induced hepatocyte damage in common carp (Cyprinus carpio)

The present study is aiming at evaluating the hepatoprotective and antioxidant effects of Glycyrrhiza glabra extract (2.5, 5 and 10 μg/ml) on the carbon tetrachloride (CCl₄)-induced carp hepatocyte damage in vitro. Glycyrrhiza glabra extract was added to the carp primary hepatocytes before (pre-treatment), after (post-treatment) and both before and after (pre- and post-treatment) the incubation of the hepatocytes with CCl₄. CCl₄ at 8 mM in the culture medium produced significantly elevated levels of lactate dehydrogenase (LDH), glutamate oxalate transaminase (GOT), glutamate pyruvate transaminase (GPT) and malondialdehyde (MDA) and significantly reduced levels of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px). Pre-treatment (5 μg/ml) and pre- and post-treatment (5 and 10 μg/ml) of the hepatocytes with Glycyrrhiza glabra extract significantly reduced the elevated levels of LDH, GOT, GPT and MDA and increased the reduced levels of SOD and GSH-Px by CCl₄; post-treatment of the hepatocytes with Glycyrrhiza glabra extract at 5 μg/ml reduced the GPT and GOT levels and increased the GSH-Px level, but had no effect on the other parameters at all the studied concentrations. The results support the use of Glycyrrhiza glabra extract as a hepatoprotective and antioxidant agent in fish.     

Influence of partial substitution of dietary fish oil by vegetable oils on the metabolism of [1-14C] 18:3n-3 in isolated hepatocytes of European sea bass (Dicentrarchus labrax L.)

The static or declining supply of fish oil from industrial fisheries demands the search of alternatives, such as plant (vegetable) oils, for diets in expanding marine aquaculture. Vegetable oils are rich in C₁₈ polyunsaturated fatty acids but devoid of the n-3 highly unsaturated fatty acids in fish oils. Previous studies, primarily with salmonids, have shown that including vegetable oils in their diets increased hepatocyte fatty acid desaturation. In the present study, we have investigated the effects of dietary partial substitution of fish oil (FO) with rapeseed oil (RO), linseed oil (LO) and olive oil (OO) on the desaturation /elongation and, -oxidation capacities of [1-¹⁴C]18:3n-3 in isolated hepatocytes from European sea bass (Dicentrarchus labrax L.), in a simultaneous combined assay. Fish were fed during 34 weeks with diets containing 100% FO, or RO, LO and OO, each included at 60% with the balance being met by FO, with no detrimental effect upon growth or survival. The highest total desaturation rates were found in hepatocytes of fish fed FO diet (0.52±0.08 pmol/h/mg protein) and OO diet (0.43±0.09 pmol/h/mg protein), which represented 3.2% and 2.7% of total [1-¹⁴C]18:3n-3 incorporated, respectively. In contrast, lowest desaturation rates were presented by hepatocytes of fish fed LO and RO diets (0.23±0.06 and 0.14±0.05 pmol/h/mg protein, respectively) represented 1.4% and 0.9% of total [1-¹⁴C]18:3n-3 incorporated, respectively. The rates of [1-¹⁴C]18:3n-3 β-oxidized were between 11-fold and 35-fold higher than desaturation. However, no significant differences were observed among β-oxidation activities in hepatocytes of fish fed any of the diets. The present study demonstrated that the European sea bass, as a carnivorous marine fish, presented a ‘marine’ fish pattern in the metabolism of 18:3n-3 to 20:5n-3 and 22:6n-3. This species appeared to have all the enzymic activities necessary to produce 22:6n-3 but presented only extremely low rates of fatty acid bioconversion. Furthermore, nutritional regulation of hepatocyte fatty acid desaturation was minimal, and dietary vegetable oils did not increase desaturase activities, and in RO and LO treatments the activity was significantly lower. This revised version was published online in July 2006 with corrections to the Cover Date.     

Purification and characterization of phospholipase A2 isoforms from the hepatopancreas of red sea bream, Pagrus major1

Two phospholipase A₂ (PLA₂) isoforms, tentatively denoted as DE-1 and DE-2 PLA₂s, were purified from the hepatopancreas of red sea bream (Pagrus major) to near homogeneity by sequential column chromatography on S-Sepharose fast flow, DEAE-Sepharose fast flow and butyl-Cellulofine, and by ion-exchange, gel-filtration and reversed-phase HPLC. The purified DE-1 and DE-2 PLA₂s both showed a single band with the apparent molecular mass of approx. 13.5 kDa by SDS-polyacrylamide gel electrophoresis, and were found to be both related to group I PLA₂ based on the N-terminal amino acid sequences. DE-1 PLA₂ had a pH optimum in the alkaline region at around pH 10 and required approximately 10 mM of Ca²⁺ and 4-10 mM of sodium deoxycholate for its maximal activity, using 2 mM of phosphatidylcholine and phosphatidylethanolamine as substrates. DE-2 PLA₂ also had a pH optimum in the alkaline region at around pH 8-9 and required >10 mM of Ca²⁺ and approximately 6 mM of sodium deoxycholate for its maximum activity with 2 mM of phosphatidylcholine as a substrate; its enzymatic activity towards phosphatidylethanolamine was greatly inhibited by the addition of sodium deoxycholate. The results demonstrate that red sea bream hepatopancreas contains two enzymatically distinct group I PLA₂ isoforms.     

Oxidative damage repair by glutamine in fish enterocytes

Fish intestine is very sensitive to oxidative damage. Repair of damaged enterocytes may be involved to restore normal function of fish intestine. However, studies of fish enterocyte repair are scarce. The present study aimed to investigate the potential repair role of glutamine after a H₂O₂ challenge. In this study, fish enterocytes were post-treated with graded levels of glutamine (0, 4, 8, 12 and 20 mM of glutamine) after expose to 100 μM H₂O₂. The basal control cells were kept in the glutamine-free minimum essential medium only. Results showed that the H₂O₂-induced decreases in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide optical density, alkaline phosphatase and Na⁺, K⁺-ATPase activities were completely restored by subsequent glutamine treatments. In addition, cellular injury (lactate dehydrogenase), lipid peroxidation (malondialdehyde) and protein oxidation (protein carbonyls) caused by H₂O₂ were reversed by subsequent glutamine treatments. Furthermore, the H₂O₂-induced decreases in glutathione contents, glutathione reductase, superoxide dismutase and glutathione peroxidase activities were completely restored by subsequent glutamine treatments. In summary, the present study indicated that glutamine improved the repair activity in fish enterocytes after challenge with H₂O₂.     

Antioxidative role of cerium against the toxicity of lead in the liver of silver crucian carp

The antioxidative role of cerium was investigated in the liver of silver crucian carp injected with lead. The fish were intraperitoneally injected with 10, 20, or 30 mg/kg wet weight PbCl₂. After a 14-day period of incubation, 35 animals were injected with a solution of 1.5 mg/kg wet weight CeCl₃. After 42 days, the wet weight and the liver weight of the fish were weighed, and the oxidative stress of the fish liver was estimated by assaying lipid peroxide, superoxide dismutase, catalase, ascorbate peroxidase, glutathione peroxidase, glutathione, ascorbic acid, and reactive oxygen species (ROS). The results show that Ce³⁺ could decrease ROS accumulation, relieve the inhibition of the activities of the antioxidant enzyme and the reduction of antioxidants in fish liver caused by Pb²⁺, and decrease the enhancement of hepatosomatic index of fish under various Pb²⁺ dosages.     

Viscera of Labeo rohita: A Potential Source of Trypsin for Industrial Application

ABSTRACT

The serine protease trypsin was isolated and purified from the digestive system of carp Labeo rohita rohu by ammonium sulphate precipitation, ion exchange, and affinity chromatography. The purified enzyme showed high activity between pH 7.0 and 9.0. The activity was maximum at 40°C. Incubation of the purified enzyme with CaCl₂ (2 mM) stabilized the enzyme activity for 8 h. The enzyme showed stability at 30 and 40°C for 1 h, but above 40°C, enzyme activity was reduced. The kinetic constants were recorded as K_m (0.104 mM), k_cat (44.25 s^?1), and catalytic efficiency (427.54 s^?1 mM^?1). Monovalent, bivalent, and trivalent ions (Li⁺, K⁺, Hg²⁺, Al³⁺, Mg^2+, Cd^2+, Co^2+, Zn²⁺, and Al³⁺) influenced the enzyme activity. Phenylmethylsulfonylflouride, soybean trypsin inhibitor, and N-α-p-tosyl-_L-lysine chloromethyl ketone completely inhibited the enzyme activity, while ethylenediaminetetraacetate caused partial inhibition. Molecular mass of the purified enzyme was 22.46 kDa. The pH and temperature stability of enzyme may be useful for its industrial applications.     

Purification and characterization of phospholipase A2 from the pyloric caeca of red sea bream, Pagrus major

A phospholipase A₂ was purified 55,000-fold in a yield of 10% from the lipid-free extract of powder of the pyloric caeca of red sea bream to near homogeneity by sequential column chromatography on S-sepharose fast flow, butyl-cellulofine, Asahipak ES-502C cation-exchange HPLC, TSK gel G3000SW gel-filtration HPLC, and Asahipak ODP-50 reversed-phase HPLC. The final preparation showed a single band with the apparent molecular mass of 14 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and an estimated specific activity was 717 µmol min^-1 mg^-1 protein. The purified enzyme had a pH optimum in the range of pH 8.0–9.0 and required the presence of both 8 mM of Ca²⁺ and from 2 to 10 mM of sodium deoxycholate for its maximal activity, using 2 mM of phosphatidylcholine as a substrate. The purified enzyme preferentially hydrolyzed the 2-acyl ester bonds of both phosphatidylglycerol and phosphatidylcholine in the presence of sodium deoxycholate, followed in order by phosphatidylethanolamine and phosphatidyl-serine. In contrast to porcine pancreatic PLA₂, pyloric caeca PLA₂ hydrolyzed mixed-micellar phosphatidylcholine substrate effectively, regardless of the kinds of bile salts used. These results indicate that Ca²⁺-dependent low molecular mass PLA₂, so called secretory PLA₂, occurs in the pyloric caeca of red sea beam.     

Effect of dietary oxidized fish oil on growth performance,body composition,antioxidant defence mechanism and liver histology of juvenile largemouth bass Micropterus salmoides

Four isonitrogenous and isolipidic diets containing fresh fish oil (peroxide value, POV: 11.5 meq kg^?1, diet FR) and three degrees of oxidized fish oil (POV: 132, 277 and 555 meq kg^?1, diet OX₁₃₂, OX₂₇₇ and OX₅₅₅, respectively) were formulated to investigate the effects of dietary oxidized fish oil on growth performance, body composition, antioxidant defence mechanism and liver histology of juvenile largemouth bass. After a 12‐week feeding trail, a proportion of approximately 9% of Micropterus salmoides showed inflammation and haemorrhage at the base of dorsal, pectoral and tail fin in both groups OX₂₇₇ and OX₅₅₅. Fish fed oxidized oil diets obtained significantly higher (P < 0.05) weight gain and specific growth rate because of their remarkable higher feed intakes, compared with the fresh oil receiving group. The analysis of biometric parameters and body composition indicated significant differences (P < 0.05) in various test diets. The activities of hepatic catalase and superoxide dismutase were significantly stimulated (P < 0.05) by oxidized oil ingestion. Hepatic glutathione peroxidase, glutathione reductase and glutathione‐S‐transferase activities were significantly higher (P < 0.05), and liver glutathione content was markedly lower (P < 0.05) in group OX₅₅₅ than the other treatments. Oxidized oil consumption resulted in marked depletion (P < 0.05) of vitamin E concentration in plasma, liver and muscle tissue, increased plasma and muscle malondialdehyde content along with decreased haematocrit value. Histological examinations indicated that hepatocytes with lipid vacuoles and nuclear migration were shown in groups OX₂₇₇ and OX₅₅₅. The overall results in this study suggested that an increased oxidative stress in M. salmoides fed oxidized lipid may account for their stimulated hepatic antioxidant defences, vitamin E depletion in plasma and certain tissues, and pathological changes. The detrimental effect of oxidation products on fish health and the unexpectedly enhanced feed intake of oxidized feeds in M. salmoides underline the importance that cares should be taken to minimize dietary oxidation products to the greatest extent possible.     

Binding and bioactivity of insulin in primary cultures of carp (Cyprinus carpio) hepatocytes

Isolated carp hepatocytes cultured in serum-free, chemically defined medium were used to investigate within the same cell preparation characteristics of the binding of insulin as well as effects of insulin on cellular metabolism. The binding of human [¹²⁵I]-insulin to carp hepatocytes was studied in kinetic, saturation and displacement experiments. A dependency of insulin binding on the collagenase used for cell isolation was demonstrated. Insulin binding decreased during the first 12h of culture but remained constant during the following 12h. The kinetic experiments revealed that [¹²⁵I]-insulin binding reached a steady state within 20–30 min of incubation. The mathematical analysis of the saturation experiments demonstrated the existence of two populations of binding sites, one with high affinity (K_d1 = 5.5 pM) and low capacity (B_max1 = 0.14 fmol/mg protein or 77 binding sites/cell) and one with low affinity (K_d2 = 2.4 nM) and high capacity (B_max2 = 17.6 fmol/mg protein or 9623 binding sites/cell). In competition experiments, 312 pM [¹²⁵I]-insulin was displaced by cold insulin, IGF-I and IGF-II with IC₅₀ values of 2.2, 7.9 and 20.3 nM, respectively. Glucagon was without effect. Binding of insulin to carp hepatocytes resulted in a significant reduction of glucose release and a significant increase of protein synthesis as of de novo fatty acid synthesis. dedicated to Prof. Dr. W. Hanke on the occasion of his 65^th birthday.     

In vitro and in vivo hepatoprotective and antioxidant effects of Astragalus polysaccharides against carbon tetrachloride-induced hepatocyte damage in common carp (Cyprinus carpio)

The present study is aiming at evaluating the hepatoprotective and antioxidant effects of Astragalus polysaccharide (APS) on the carbon tetrachloride (CCl₄)-induced hepatocyte and liver injury in common carp in vitro and in vivo. In vitro, APS (200, 400 and 800 μg/ml) was added to the carp primary hepatocytes before (pre-treatment), after (post-treatment) and both before and after (pre- and post-treatment) the incubation of the hepatocytes with CCl₄ at 8 mM in the culture medium. APS at concentrations of 200, 400 and 800 μg/ml significantly improved cell viability and inhibited the elevation of glutamate pyruvate transaminase (GPT), glutamate oxalate transaminase (GOT), lactate dehydrogenase (LDH) and malondialdehyde (MDA) and significantly increased the reduced level of superoxide dismutase (SOD). In vivo administration of APS at the doses of 1.5 and 3 g/kg in the diet for 60 days prior to CCl₄ intoxication significantly reduced the elevated activities of GPT, GOT and LDH and increased the reduced levels of total protein and albumin in the serum; meanwhile, the reduced levels of SOD, glutathione and total antioxidant capacity (T-AOC) were markedly increased and the MDA formation was significantly inhibited in liver tissue. Overall results proved the hepatoprotective action of APS, which is likely related to its antioxidant activity. The results support the use of APS as a hepatoprotective and antioxidant agent in fish.     

Influence of cell volume changes on protein synthesis in isolated hepatocytes of air-breathing walking catfish (<Emphasis Type="Italic">Clarias batrachus</Emphasis>)

The present study aimed at determining the effect of cell volume changes on protein synthesis, measured as the incorporation of [³H]leucine into acid-precipitable protein, in isolated hepatocytes of air-breathing walking catfish (Clarias batrachus). The rate of protein synthesis, which was recorded to be 10.02 ± 0.10 (n = 25) nmoles mg⁻¹ cell protein h⁻¹ in isotonic incubation conditions, increased/decreased significantly by 18 and 48%, respectively, following hypo- (−80 mOsmol l⁻¹)/hypertonic (+80 mOsmol l⁻¹) incubation conditions (adjusted with NaCl), with an accompanying increase/decrease of hepatic cell volume by 12 and 20%, respectively. Similar cell volume-sensitive changes of protein synthesis were also observed when the anisotonicity of incubation medium was adjusted with mannitol. Increase of hepatic cell volume by 9%, due to addition of glutamine plus glycine (5 mM each) to the isotonic control incubation medium, led to a significant increase of protein synthesis by 14%. Decrease of hepatic cell volume by 15 and 18%, due to addition of dibutyl-cAMP and adenosine in isotonic control incubation medium, led to a significant decrease of protein synthesis by 30 and 34%, respectively. Thus, it appears that the increase/decrease of hepatic cell volume, caused either by changing the extracellular osmolarity or by the presence of amino acids or certain other metabolites, leads to increase/decrease of protein synthesis, respectively, and shows a direct correction (r = 0.99) between the hepatic cell volume and protein synthesis in walking catfish. These cell volume-sensitive changes of protein synthesis probably help this walking catfish in fine tuning the different metabolic pathways for better adaptation during cell volume changes and also to avoid the adverse affects of osmotic stress. This is the first report of cell volume-sensitive changes of protein synthesis in hepatic cells of any teleosts.     

Comparative changes in water quality and role of pond soil after application of different levels of organic and inorganic inputs

Changes in water parameters were studied in a yard experiment for 7 weeks after application of cow dung at 20, 50 and 100 t ha^?1, poultry manure at 4, 10 and 20 t ha^?1, feed mixture (groundnut oil cake and rice bran at 1:1) at 10, 20 and 30 t ha^?1 and inorganic fertilizers at 100 kg urea+50 kg single super phosphate (SSP) ha^?1, twice this dose (2x ha^?1) and thrice this dose (3x ha^?1). To study the role of soil in the mineralization process, each treatment was divided into two groups – one with and the other without soil substrate. Higher degree of changes in water parameters was observed at higher input levels. Both organic amendment and inorganic fertilization caused significant reduction (P<0.05) in dissolved oxygen and increase in free CO₂, dissolved organic matter, total ammonia, nitrite, nitrate and phosphorus contents of water. Organic inputs significantly decreased (P<0.05) water pH and increased total alkalinity and hardness. In contrast, inorganic fertilization caused a significant increase in pH; alkalinity and hardness increased significantly in the presence of soil, but reduced in its absence. In organic input, presence of soil substrate caused significantly lower value of pH, dissolved oxygen, dissolved organic matter and phosphate‐phosphorus and significantly higher free CO₂, alkalinity, hardness, ammonia, nitrite and nitrate contents, compared with those in the absence of soil, revealing enhanced microbial mineralization in the presence of soil.     

Nutritional and environmental regulation of the synthesis of highly unsaturated fatty acids and of fatty-acid oxidation in Atlantic salmon (Salmo salar L.) enterocytes and hepatocytes

The objective of this work was to determine whether highly unsaturated fatty acid (HUFA) synthesis and fatty-acid oxidation in Atlantic salmon (Salmo salar L.) intestine was under environmental and/or seasonal regulation. Triplicate groups of salmon were grown through a full two-year cycle on two diets containing either fish oil (FO) or a diet with 75% of the FO replaced by a vegetable oil (VO) blend containing rapeseed, palm, and linseed oils. At key points in the life cycle fatty acyl desaturation/elongation (HUFA synthesis) and oxidation activity were determined in enterocytes and hepatocytes using [1−¹⁴C]18:3n−3 as substrate. As observed previously, HUFA synthesis in hepatocytes reached a peak at seawater transfer and declined thereafter, with activity consistently greater in fish fed the VO diet. In fish fed FO, HUFA synthesis in enterocytes in the freshwater stage was at a level similar to that in hepatocytes. HUFA synthesis in enterocytes increased rapidly after seawater transfer, however, and remained high for some months after transfer before decreasing to levels that were again similar to those observed in hepatocytes. Enterocyte synthesis of HUFA was usually higher in fish fed the VO diet than in those fed the FO diet. Oxidation of [1−¹⁴C]18:3n−3 in hepatocytes from fish fed FO tended to decrease during the freshwater phase but then increased steeply, peaking just after transfer before decreasing during the remaining seawater phase. At the peak in oxidation activity around seawater transfer, activity was significantly lower in fish fed VO than in fish fed FO. In enterocytes, oxidation of [1−¹⁴C]18:3 in fish fed FO reached a peak in activity just before seawater transfer. In fish fed VO, except for high activity at nine months the pattern was similar to that obtained in enterocytes from fish fed FO, with high activity around seawater transfer and declining activity in seawater. In conclusion, fatty acid metabolism in intestinal cells seemed to be under dual nutritional and environmental or seasonal regulation. Temporal patterns of oxidation of fatty acids were usually similar in the two cell types, but HUFA synthesis in enterocytes peaked over the summer seawater phase rather than at transfer, as with hepatocytes, suggesting the possibility of different regulatory cues.     

Muscle ammonia stores are not determined by pH gradients

The theory of non-ionic diffusion predicts that ammonia will distribute between intracellular and extracellular tissue compartments according to transmembrane pH gradients. The distribution of ammonia and¹⁴C-DMO were compared in white muscle and plasma of rainbow trout (Salmo gairdneri) at rest, and following exhaustive exercise. Under both experimental conditions, intracellular ammonia levels far exceeded those predicted by transmembrane pH gradients. Calculated equilibrium potentials for ) were very close to published resting values of membrane potential E_m in fish white muscle. We conclude that NH ₄ ⁺ is permeable across cell membranes and that intracellular ammonia stores are not determined by pH gradients.The term ammonia or T_amm will be used to indicate the total ammonia concentration, while NH ₄ ⁺ and NH₃ will refer to ammonium ion and nonionic ammonia, respectively.